RESUMEN
Phosphoryl transfer is a fundamental reaction in cellular signaling and metabolism that requires Mg2+ as an essential cofactor. While the primary function of Mg2+ is electrostatic activation of substrates, such as ATP, the full spectrum of catalytic mechanisms exerted by Mg2+ is not known. In this study, we integrate structural biology methods, molecular dynamic (MD) simulations, phylogeny, and enzymology assays to provide molecular insights into Mg2+-dependent structural reorganization in the active site of the metabolic enzyme adenylate kinase. Our results demonstrate that Mg2+ induces a conformational rearrangement of the substrates (ATP and ADP), resulting in a 30° adjustment of the angle essential for reversible phosphoryl transfer, thereby optimizing it for catalysis. MD simulations revealed transitions between conformational substates that link the fluctuation of the angle to large-scale enzyme dynamics. The findings contribute detailed insight into Mg2+ activation of enzymes and may be relevant for reversible and irreversible phosphoryl transfer reactions.
Asunto(s)
Adenilato Quinasa , Dominio Catalítico , Magnesio , Simulación de Dinámica Molecular , Magnesio/metabolismo , Magnesio/química , Adenilato Quinasa/metabolismo , Adenilato Quinasa/química , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Conformación Proteica , Adenosina Difosfato/metabolismo , Adenosina Difosfato/químicaRESUMEN
Understanding enzyme mechanisms is essential for unraveling the complex molecular machinery of life. In this review, we survey the field of computational enzymology, highlighting key principles governing enzyme mechanisms and discussing ongoing challenges and promising advances. Over the years, computer simulations have become indispensable in the study of enzyme mechanisms, with the integration of experimental and computational exploration now established as a holistic approach to gain deep insights into enzymatic catalysis. Numerous studies have demonstrated the power of computer simulations in characterizing reaction pathways, transition states, substrate selectivity, product distribution, and dynamic conformational changes for various enzymes. Nevertheless, significant challenges remain in investigating the mechanisms of complex multistep reactions, large-scale conformational changes, and allosteric regulation. Beyond mechanistic studies, computational enzyme modeling has emerged as an essential tool for computer-aided enzyme design and the rational discovery of covalent drugs for targeted therapies. Overall, enzyme design/engineering and covalent drug development can greatly benefit from our understanding of the detailed mechanisms of enzymes, such as protein dynamics, entropy contributions, and allostery, as revealed by computational studies. Such a convergence of different research approaches is expected to continue, creating synergies in enzyme research. This review, by outlining the ever-expanding field of enzyme research, aims to provide guidance for future research directions and facilitate new developments in this important and evolving field.
RESUMEN
This study explores ligand-driven conformational changes in adenylate kinase (AK), which is known for its open-to-close conformational transitions upon ligand binding and release. By utilizing string free energy simulations, we determine the free energy profiles for both enzyme opening and ligand release and compare them with profiles from the apoenzyme. Results reveal a three-step ligand release process, which initiates with the opening of the adenosine triphosphate-binding subdomain (ATP lid), followed by ligand release and concomitant opening of the adenosine monophosphate-binding subdomain (AMP lid). The ligands then transition to nonspecific positions before complete dissociation. In these processes, the first step is energetically driven by ATP lid opening, whereas the second step is driven by ATP release. In contrast, the AMP lid opening and its ligand release make minor contributions to the total free energy for enzyme opening. Regarding the ligand binding mechanism, our results suggest that AMP lid closure occurs via an induced-fit mechanism triggered by AMP binding, whereas ATP lid closure follows conformational selection. This difference in the closure mechanisms provides an explanation with implications for the debate on ligand-driven conformational changes of AK. Additionally, we determine an X-ray structure of an AK variant that exhibits significant rearrangements in the stacking of catalytic arginines, explaining its reduced catalytic activity. In the context of apoenzyme opening, the sequence of events is different. Here, the AMP lid opens first while the ATP lid remains closed, and the free energy associated with ATP lid opening varies with orientation, aligning with the reported AK opening and closing rate heterogeneity. Finally, this study, in conjunction with our previous research, provides a comprehensive view of the intricate interplay between various structural elements, ligands, and catalytic residues that collectively contribute to the robust catalytic power of the enzyme.
Asunto(s)
Adenosina Trifosfato , Adenilato Quinasa , Adenilato Quinasa/química , Ligandos , Apoenzimas/metabolismo , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Conformación ProteicaRESUMEN
Adenylate kinases play a crucial role in cellular energy homeostasis through the interconversion of ATP, AMP, and ADP in all living organisms. Here, we explore how adenylate kinase (AdK) from Escherichia coli interacts with diadenosine tetraphosphate (AP4A), a putative alarmone associated with transcriptional regulation, stress, and DNA damage response. From a combination of EPR and NMR spectroscopy together with X-ray crystallography, we found that AdK interacts with AP4A with two distinct modes that occur on disparate time scales. First, AdK dynamically interconverts between open and closed states with equal weights in the presence of AP4A. On a much slower time scale, AdK hydrolyses AP4A, and we suggest that the dynamically accessed substrate-bound open AdK conformation enables this hydrolytic activity. The partitioning of the enzyme into open and closed states is discussed in relation to a recently proposed linkage between active site dynamics and collective conformational dynamics.
Asunto(s)
Adenilato Quinasa , Escherichia coli , Escherichia coli/metabolismo , Adenilato Quinasa/química , Hidrólisis , Fosfatos de Dinucleósidos/metabolismo , Catálisis , Dominio CatalíticoRESUMEN
Biological life depends on motion, and this manifests itself in proteins that display motion over a formidable range of time scales spanning from femtoseconds vibrations of atoms at enzymatic transition states, all the way to slow domain motions occurring on micro to milliseconds. An outstanding challenge in contemporary biophysics and structural biology is a quantitative understanding of the linkages among protein structure, dynamics, and function. These linkages are becoming increasingly explorable due to conceptual and methodological advances. In this Perspective article, we will point toward future directions of the field of protein dynamics with an emphasis on enzymes. Research questions in the field are becoming increasingly complex such as the mechanistic understanding of high-order interaction networks in allosteric signal propagation through a protein matrix, or the connection between local and collective motions. In analogy to the solution to the "protein folding problem," we argue that the way forward to understanding these and other important questions lies in the successful integration of experiment and computation, while utilizing the present rapid expansion of sequence and structure space. Looking forward, the future is bright, and we are in a period where we are on the doorstep to, at least in part, comprehend the importance of dynamics for biological function.
RESUMEN
Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme's conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme's functionâone to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK's conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme's conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme's active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.
Asunto(s)
Adenilato Quinasa , Simulación de Dinámica Molecular , Adenilato Quinasa/química , Catálisis , Dominio Catalítico , Escherichia coli/metabolismo , Conformación ProteicaRESUMEN
Enzymatic catalysis is critically dependent on selectivity, active site architecture, and dynamics. To contribute insights into the interplay of these properties, we established an approach with NMR, crystallography, and MD simulations focused on the ubiquitous phosphotransferase adenylate kinase (AK) isolated from Odinarchaeota (OdinAK). Odinarchaeota belongs to the Asgard archaeal phylum that is believed to be the closest known ancestor to eukaryotes. We show that OdinAK is a hyperthermophilic trimer that, contrary to other AK family members, can use all NTPs for its phosphorylation reaction. Crystallographic structures of OdinAK-NTP complexes revealed a universal NTP-binding motif, while 19F NMR experiments uncovered a conserved and rate-limiting dynamic signature. As a consequence of trimerization, the active site of OdinAK was found to be lacking a critical catalytic residue and is therefore considered to be "atypical." On the basis of discovered relationships with human monomeric homologs, our findings are discussed in terms of evolution of enzymatic substrate specificity and cold adaptation.
Asunto(s)
Adenilato Quinasa , Archaea , Humanos , Archaea/genética , Adenilato Quinasa/química , Catálisis , Dominio CatalíticoRESUMEN
The biological function of proteins is critically dependent on dynamics inherent to the native structure. Such structural dynamics obey a predefined order and temporal timing to execute the specific reaction. Determination of the cooperativity of key structural rearrangements requires monitoring protein reactions in real time. In this work, we used time-resolved x-ray solution scattering (TR-XSS) to visualize structural changes in the Escherichia coli adenylate kinase (AdK) enzyme upon laser-induced activation of a protected ATP substrate. A 4.3-ms transient intermediate showed partial closing of both the ATP- and AMP-binding domains, which indicates a cooperative closing mechanism. The ATP-binding domain also showed local unfolding and breaking of an Arg131-Asp146 salt bridge. Nuclear magnetic resonance spectroscopy data identified similar unfolding in an Arg131Ala AdK mutant, which refolded in a closed, substrate-binding conformation. The observed structural dynamics agree with a "cracking mechanism" proposed to underlie global structural transformation, such as allostery, in proteins.
RESUMEN
Enzymes employ a wide range of protein motions to achieve efficient catalysis of chemical reactions. While the role of collective protein motions in substrate binding, product release, and regulation of enzymatic activity is generally understood, their roles in catalytic steps per se remain uncertain. Here, molecular dynamics simulations, enzyme kinetics, X-ray crystallography, and nuclear magnetic resonance spectroscopy are combined to elucidate the catalytic mechanism of adenylate kinase and to delineate the roles of catalytic residues in catalysis and the conformational change in the enzyme. This study reveals that the motions in the active site, which occur on a time scale of picoseconds to nanoseconds, link the catalytic reaction to the slow conformational dynamics of the enzyme by modulating the free energy landscapes of subdomain motions. In particular, substantial conformational rearrangement occurs in the active site following the catalytic reaction. This rearrangement not only affects the reaction barrier but also promotes a more open conformation of the enzyme after the reaction, which then results in an accelerated opening of the enzyme compared to that of the reactant state. The results illustrate a linkage between enzymatic catalysis and collective protein motions, whereby the disparate time scales between the two processes are bridged by a cascade of intermediate-scale motion of catalytic residues modulating the free energy landscapes of the catalytic and conformational change processes.
Asunto(s)
Adenilato Quinasa/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/química , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E.coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.
Asunto(s)
Bombyx , Escherichia coli/genética , Proteínas de Insectos , Proteínas Recombinantes , Serina Proteasas , Animales , Bombyx/enzimología , Bombyx/genética , Escherichia coli/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/metabolismo , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serina Proteasas/química , Serina Proteasas/genética , Serina Proteasas/aislamiento & purificación , Serina Proteasas/metabolismoRESUMEN
ATP and GTP are exceptionally important molecules in biology with multiple, and often discrete, functions. Therefore, enzymes that bind to either of them must develop robust mechanisms to selectively utilize one or the other. Here, this specific problem is addressed by molecular studies of the human NMP kinase AK3, which uses GTP to phosphorylate AMP. AK3 plays an important role in the citric acid cycle, where it is responsible for GTP/GDP recycling. By combining a structural biology approach with functional experiments, we present a comprehensive structural and mechanistic understanding of the enzyme. We discovered that AK3 functions by recruitment of GTP to the active site, while ATP is rejected and nonproductively bound to the AMP binding site. Consequently, ATP acts as an inhibitor with respect to GTP and AMP. The overall features with specific recognition of the correct substrate and nonproductive binding by the incorrect substrate bear a strong similarity to previous findings for the ATP specific NMP kinase adenylate kinase. Taken together, we are now able to provide the fundamental principles for GTP and ATP selectivity in the large NMP kinase family. As a side-result originating from nonlinearity of chemical shifts in GTP and ATP titrations, we find that protein surfaces offer a general and weak binding affinity for both GTP and ATP. These nonspecific interactions likely act to lower the available intracellular GTP and ATP concentrations and may have driven evolution of the Michaelis constants of NMP kinases accordingly.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Guanosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Adenilato Quinasa/química , Biocatálisis , Guanosina Trifosfato/química , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Especificidad por SustratoRESUMEN
As a key molecule in biology, adenosine triphosphate (ATP) has numerous crucial functions in, for instance, energetics, post-translational modifications, nucleotide biosynthesis, and cofactor metabolism. Here, we have discovered an intricate interplay between the enzyme adenylate kinase and its substrate ATP. The side chain of an arginine residue was found to be an efficient sensor of the aromatic moiety of ATP through the formation of a strong cation-π interaction. In addition to recognition, the interaction was found to have dual functionality. First, it nucleates the activating conformational transition of the ATP binding domain and also affects the specificity in the distant AMP binding domain. In light of the functional consequences resulting from the cation-π interaction, it is possible that the mode of ATP recognition may be a useful tool in enzyme design.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adenilato Quinasa/metabolismo , Adenosina Trifosfato/química , Adenilato Quinasa/química , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their ß-barrel domain in response to the fluctuating acidity of the thylakoid lumen.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Spinacia oleracea/metabolismo , Tilacoides/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.
Asunto(s)
Proteínas Bacterianas/química , Sistemas de Secreción Bacterianos , Complejos Multiproteicos/química , Proteínas Bacterianas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Relación Estructura-Actividad , Yersinia pseudotuberculosis/metabolismoRESUMEN
Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.
Asunto(s)
Adenosina Trifosfato/metabolismo , Adenilil Ciclasas/metabolismo , Guanosina Trifosfato/metabolismo , Inhibidores de Adenilato Ciclasa/química , Inhibidores de Adenilato Ciclasa/farmacología , Inosina Trifosfato/genética , Inosina Trifosfato/metabolismo , Cinética , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Proteins can bind target molecules through either induced fit or conformational selection pathways. In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation. In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover. The transient nature of these states has precluded direct observation of their properties. Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography. Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis. A 1.9-Å X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis. We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state. In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance. Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change.
Asunto(s)
Adenilato Quinasa/química , Adenilato Quinasa/metabolismo , Adenilato Quinasa/genética , Sustitución de Aminoácidos , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/química , Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , TermodinámicaRESUMEN
Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia, the switch to secretion of effector proteins is induced first after intimate contact between the bacterium and its eukaryotic target cell has been established, and the T3SS proteins YscP and YscU play a central role in this process. Here we identify the molecular details of the YscP binding site on YscU by means of nuclear magnetic resonance (NMR) spectroscopy. The binding interface is centered on the C-terminal domain of YscU. Disrupting the YscU-YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the binding of YscP to the slowly self-cleaving YscU variant P264A conferred significant protection against autoproteolysis. The YscP-mediated inhibition of YscU autoproteolysis suggests that the cleavage event may act as a timing switch in the regulation of early versus late T3SS substrates. We also show that YscUC binds to the inner rod protein YscI with a dissociation constant (Kd ) of 3.8 µm and with 1:1 stoichiometry. The significant similarity among different members of the YscU, YscP, and YscI families suggests that the protein-protein interactions discussed in this study are also relevant for other T3SS-containing Gram-negative bacteria.
Asunto(s)
Mapas de Interacción de Proteínas , Sistemas de Secreción Tipo III/metabolismo , Infecciones por Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Especificidad por Sustrato , Sistemas de Secreción Tipo III/química , Yersinia pseudotuberculosis/química , Infecciones por Yersinia pseudotuberculosis/microbiologíaRESUMEN
Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a 31P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states.
Asunto(s)
Adenilato Quinasa/química , Urea/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Animales , Dicroismo Circular , Estabilidad de Enzimas , Escherichia coli , Cinética , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Músculos/química , Músculos/enzimología , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Desplegamiento Proteico , Conejos , Termodinámica , Urea/metabolismo , Agua/químicaRESUMEN
Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies.
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Adenilato Quinasa/metabolismo , Ligamiento Genético , Levaduras/metabolismo , Adenilato Quinasa/genética , Catálisis , Plásmidos , Levaduras/enzimología , Levaduras/genéticaRESUMEN
It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (µs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.